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Open AcceResearch articleTransmission potential, skin inflammatory response, and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasisBLA Verçosa1, CM Lemos1, IL Mendonça1, SMMS Silva1, SM de Carvalho1, H Goto2 and FAL Costa*1

Address: 1Departamento de Clinica e Cirurgia Veterinária, Centro de Ciências Agrárias, Universidade Federal do Piauí, Teresina-Pi, Brasil and 2Departamento de Medicina Preventiva, Faculdade de Medicina, Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo-SP, Brasil

Email: BLA Verçosa - brbaravet@yahoo.com.br; CM Lemos - cristin_lemos@zipmail.com.br; IL Mendonça - ivetemendonca@ig.com.br; SMMS Silva - silvanammss@ufpi.br; SM de Carvalho - soniacarvalho@click21.com.br; H Goto - hgoto@usp.br; FAL Costa* - fassisle@gmail.com

* Corresponding author

AbstractBackground: Visceral leishmaniasis in Brazil is caused by the protozoan Leishmania (Leishmania) chagasi and it istransmitted by sandfly of the genus Lutzomyia. Dogs are an important domestic reservoir, and control of thetransmission of visceral leishmaniasis (VL) to humans includes the elimination of infected dogs. However, thoughdogs are considered to be an important element in the transmission cycle of Leishmania, the identification ofinfected dogs representing an immediate risk for transmission has not been properly evaluated. Since it is notpossible to treat infected dogs, they are sacrificed when a diagnosis of VL is established, a measure that is difficultto accomplish in highly endemic areas. In such areas, parameters that allow for easy identification of reservoirsthat represents an immediate risk for transmission is of great importance for the control of VL transmission. Inthis study we aimed to identify clinical parameters, reinforced by pathological parameters that characterize dogswith potential to transmit the parasite to the vector.

Results: The major clinical manifestations of visceral leishmaniasis in dogs from an endemic area wereonicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. The transmission potential of thesedogs was assessed by xenodiagnosis using Lutzomyia longipalpis. Six of nine symptomatic dogs were infective toLutzomyia longipalpis while none of the five asymptomatic dogs were infective to the sandfly. Leishmaniaamastigotes were present in the skin of all clinically symptomatic dogs, but absent in asymptomatic dogs. Higherparasite loads were observed in the ear and ungueal region, and lower in abdomen. The inflammatory infiltratewas more intense in the ears and ungueal regions of both symptomatic and asymptomatic dogs. In clinicallyaffected dogs in which few or none Leishmania amastigotes were observed, the inflammatory infiltrate wasconstituted mainly of lymphocytes and macrophages. When many parasites were present, the infiltrate was alsocomprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils(PMNs).

Conclusion: Dogs that represent an immediate risk for transmission of Leishmania in endemic areas presentclinical manifestations that include onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss.Lymphadenopathy in particular was a positive clinical hallmark since it was closely related to the positivexenodiagnosis.

Published: 6 November 2008

BMC Veterinary Research 2008, 4:45 doi:10.1186/1746-6148-4-45

Received: 27 February 2008Accepted: 6 November 2008

This article is available from: http://www.biomedcentral.com/1746-6148/4/45

© 2008 Verçosa et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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BackgroundVisceral leishmaniasis (VL) in Brazil is caused by Leishma-nia (Leishmania) chagasi and it is transmitted by the sand-fly Lutzomyia longipalpis [1]. The dog is considered to bethe main domestic reservoir of Leishmania chagasi becauseit presents intense parasitism in the skin, allowing for easytransmission of Leishmania to the sandfly [2-4]. Therefore,dogs have been the target of control measures for thetransmission of Leishmania to humans. In this context, theidentification of infected dogs that represent an immedi-ate risk for transmission is of utmost importance, a pointthat deserves meticulous study. In this study we analyzedclinical manifestation as a parameter that may indicatethe immediate risk for transmission.

In endemic areas, although 67% to 80% of the animalshave contact with the parasite as demonstrated either bythe presence of anti-Leishmania antibodies or by specificcell-mediated immune response or by detection of Leish-mania-related polymerase chain reaction products, manyhave no signs of disease [5-7]. In addition, symptoms sug-gestive of other diseases are also observed in some dogs,blurring the diagnosis of VL [8,9]. Since it is not possibleto treat infected dogs, they are all sacrificed when the diag-nosis of VL is established, a measure that is difficult toaccomplish mainly in highly endemic areas. It is knownthat only some infected dogs effectively transmit the dis-ease, that skin parasitism of dogs does not occur at thesame intensity in all phases of the infection, and seem-ingly it does not correlate to the transmission potential tovectors [4,10,11]. Concerning transmission potential ofsymptomatic and asymptomatic dogs, data in the litera-ture are controversial may be due to Leishmania speciesand geographic differences. A study carried out in Spainhas shown no correlation whilst those in Colombia andBrazil have shown a positive correlation of the presence ofsymptoms to the infectivity [4,12,13]. In experimental L.chagasi or L. donovani-infected dogs infection of the vectorwas more likely to occur when fed on dogs at moreadvanced stage of the disease [14]. Therefore, the presentstudy was aimed at identifying clinical parameters, rein-forced by pathological parameters, that characterize dogswith potential to transmit the parasite to the vector in anendemic area in Brazil. We examined clinical presenta-tions, the skin inflammatory process, parasite load andtransmission potential by xenodiagnosis in dogs with vis-ceral leishmaniasis.

MethodsAnimals and VL diagnosisTwenty eight dogs of this study included both privatelyowned and stray dogs of the endemic area of Teresina,State of Piaui, in Brazil. Male and female adult dogs of dif-ferent ages and breeds were randomly tested for leishma-niasis by serology (mandatory in areas endemic forleishmaniasis) in epidemiological survey performed by

the Center for Zoonosis Control. The diagnosis of VL wasconfirmed by a positive anti-Leishmania serology com-bined with the detection of the parasite. For the detectionof anti-Leishmania antibodies in the sera, an indirectimmunofluorescence assay or enzyme-linked immuno-sorbent assay were used, and for the detection of Leishma-nia we have examined directly the smears of the skin,spleen and popliteal lymph nodes stained with Giemsa,or we have performed culture of material from sternalbone marrow, spleen, and/or popliteal lymph nodes (orall) in NNN medium (SIGMA-ALDRICH). The animalswere classified in three groups: a) 12 infected dogs andwith clinical signs of disease; b) 11 infected dogs but with-out any clinical signs of VL; and c) five dogs serologicallyand parasitologically negative for VL as control group.Dogs were considered symptomatic when at least one ofthe following symptoms was present: onicogriphosis, skinlesions, loss of weight, local or generalized lymphadenop-athy, diarrhea, epistaxis, conjunctivitis, anorexia, or fever.Asymptomatic dogs were those infected but without anysymptoms of VL, and the diagnosis established by sero-logical test and positive parasitological exam. The param-eters used to classify clinically affected and non-affecteddogs were based on the classification proposed by Pozioet al. [15].

Dogs with anemia were identified by to observe themucosae color during necropsy. The ocular, oral, analand/or preputial and vaginal mucosa were seen ininfected dogs for the intensity of the color, compared withnon-infected dogs. The pale mucosae was considered asanemia taking care to discard the possibility of hypostasiscadaverous.

XenodiagnosisIn nine symptomatic and five asymptomatic dogs xenodi-agnosis was performed before their sacrifice for tissuesample harvest. Briefly, the dogs were anaesthetized with1% acepromazine (0,25 mg/kg; Acepran, Univet) and 60female Lutzomyia longipalpis sandflies were allowed to feedfor 45 minutes on the skin of the ear. Five days after feed-ing, the sandflies were dissected and the middle gutremoved to observe the presence of promastigotes.

Cytological and histopathologic analysis of the skinThe animals were sedated with 1% acepromazine (0.01ml/kg), induced with xylazin (2 mg/kg; Rompum, Bayer)and anesthetized with sodium 2.5% thiopental (0.5 ml/kg). Samples of skin were taken bilaterally from the fol-lowing places: muzzle, eyelid, ear, metacarpi, forelimbungueal region, dorsum, hind limb ungueal region, meta-tarsi, tail, abdomen, and scrotum. Their imprints werestained with Giemsa, and tissue samples were formalinfixed and paraffin embedded for histopathological andimmunohistochemical studies. After harvest, the animalswere killed with an overdose of sodium thiopental. All

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Leishmania-infected dogs were routinely sacrificed by theCenter of Zoonosis for Control of VL transmission. Thenon-infected control animals were stray dogs from thesame area that were captured and sacrificed for rabies con-trol. All procedures involving animals were performedaccording to the Brazilian guide for care and use of labo-ratory animals (Projeto de lei 3.964/97 – http://www.planalto.gov.br), and all experimental protocolsused were previously approved by the Ethics Committeeof the Federal University of Piaui.

Skin histopathological alterations were examined in allsamples from 22 different locations, and the intensity wassemi-quantified blindly by two independent observers,scored from 0 to 4, and the median score assessed whenconsidering all samples from all 22 different places wasused for comparison.

Evaluation of parasite loadThe parasite load was determined in cytological imprintsstained with Giemsa and tissue sections stained by immu-noperoxidase from all 22 different places from each ani-mal in 50 × 100 field areas using a reticule of 10 mm2.Detection of Leishmania antigen by immunohistochemis-try was performed as previously described using mousepolyclonal anti-Leishmania (Leishmania) amazonensis anti-body that was produced by the Laboratory of Soroepide-miology and Immunobiology of Tropical MedicineInstitute of University of Sao Paulo [16]. In brief, the anti-body was obtained from Leishmania (Leishmania) amazon-ensis amastigote-infected BALB/c mice, tested on formalinfixed and paraffin-embedded sections of Leishmania (L.)chagasi-infected hamster liver (positive control) and thespecificity confirmed on the same sections using serumadsorbed with Leishmania (Leishmania) amazonensis pro-mastigotes when the reaction resulted negative For thereaction it was diluted 1:1.600 (vol/vol) in PBS andreacted overnight at 4°C in a humid atmosphere.

Samples were assessed using a sensitive EnVision+, perox-idase system (Dako Corporation, Carpinteria, CA, USA,Código K 4001) following protocols provided by themanufacturer.

Statistical analysesThe data were analyzed by the Spearman's correlation ornon-parametric Fisher's exact and Kruskal-Wallis tests.Those cases with a significant difference in the latter wereanalyzed with the Student-Newman-Keuls or Dunn testfor multiple comparison of groups. We considered p <0.05 to be significant.

Results and discussionAll dogs except for the control group had the visceral leish-maniasis confirmed by serology and parasitological exam.

Detection of dogs with VL in endemic areas is difficultbecause clinical signs are very variable and frequently sim-ilar to manifestations consistent with other diseases [8,9].Our results revealed that more frequent manifestationswere onicogriphosis (83.3%), skin lesions (83.3%), con-junctivitis (75%), local or generalized lymphadenopathy(66.6%), and weight loss (58.3%) (Additional file 1).Since symptomatic dogs independent of number of signshad the diagnosis of visceral leishmaniasis confirmed, wesuggest these five clinical manifestations of dogs to beconsidered for the diagnosis of canine VL. However, in agroup of 35 dogs previously studied by us two from 16animals presenting one or two symptoms were parasito-logically negative (data not shown). Therefore we suggestthat only one or two of these symptoms are not sufficient,but three of them allow us to consider the animal as sus-pect, and five as strongly suggestive of VL. Such a clinicalcriteria may contribute to quickly identify dogs with vis-ceral leishmaniasis in endemic areas.

To analyze the transmission potential of these dogs, xeno-diagnosis was performed on the ear, where the parasiteload was observed to be higher. From the 12 clinicallyaffected dogs, xenodiagnosis was performed in nine.Among them, six resulted in transmission to Lutzomyialongipalpis, while none of the five asymptomatic dogs wereinfective to the sandfly. These data suggest that dogs thatmay represent a real threat for Leishmania transmission tothe sandfly are those symptomatic ones (p = 0.0310,Fisher's exact test) data that are corroborated by similarresults from other studies performed with Lutzomyia longi-palpis, Lutzomyia youngi and Phlebotomus perniciosus [4,11].A previous study has shown that dogs with the potentialto infect the sandfly are those with at least one of the fourmain clinical manifestations [13]. Furthermore, fiveasymptomatic dogs with no parasites, as assessed by PCRof the skin in one study, and dogs with no parasites in theskin as assessed by immunohistochemistry in anotherstudy were also unable to transmit Leishmania to the sand-fly [4,10]. On the other hand asymptomatic dogs of anendemic area in Spain have been shown to be effective totransmit Leishmania infantum to Phlebotomus perniciosus[12]. These data showing differences between studies car-ried out in Mediterranean region and South America maybe due to Leishmania strain and vector species differencessince it is known that Phlebotomus perniciosus is more effec-tive than Lutzomyia longipalpis as vector to transmit vis-cerotropic Leishmania [4].

The parasite burden in the skin was analyzed from 22 dif-ferent regions of the body of dogs with VL. Higher parasiteload was observed in the ear and ungueal region, andlower parasite load was observed in the abdomen usingmaterial stained by different methods. The median valueis presented in Additional file 2 in tissue sections stained

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by immunoperoxidase. Clinically affected dogs exhibitedamastigotes in at least one of the regions examined, andpositive regions were always associated with inflamma-tory processes. In the asymptomatic (Figure 1A) and con-trol dogs (Figure 1B), no amastigotes were present in any

of the regions examined. The parasite load was higher inclinically affected dogs when compared with the asympto-matic animals (P < 0.05, Kruskal Wallis and Student-New-man-Keuls tests) (Figure 2A).

SkinFigure 1Skin. Leishmania (L.) chagasi-naturally infected (A, C, D, E, F) and non-infected dog (B). A) Absence of amastigotes in skin of the ear of asymptomatic dog; B) Absence of amastigotes in skin of the ear of control dog. C) Presence of few amastigotes in skin with minimal inflammatory infiltrate. D) Minimal inflammatory infiltrate constituted by lymphocytes, plasma cells and macro-phages. E) Presence of many parasites in skin with severe inflammation. F) Severe inflammatory infiltrate constituted by macro-phages and neutrophils. Imunoperoxidase staining (A, B, C, E). H-E staining (D, F). Original magnifications: ×140.

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SkinFigure 2Skin. Dog naturally infected with Leishmania (L.) chagasi. A) Semi-quantitative analyses of parasite load (median scores and 25–75 percentile intervals) of clinically affected and asymptomatic and control dogs. * p < 0.05 (Kruskal Wallis and Student-New-man-Keuls tests). B) Semi-quantitative analyses of inflammatory infiltrate in the skin (median scores and 25–75 percentile inter-vals) in symptomatic, asymptomatic and non-infected control dogs. * p < 0.05 (Kruskal Wallis and Dunn tests). C) Correlation between presence of amastigotes and inflammatory infiltrate in symptomatic dogs. P < 0.05 (Spearman's test). N = number of animals per group.

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From 12 symptomatic dogs, skin lesions were observed in10 animals. However, an inflammatory infiltrate in theskin was present in all animals, even in those withoutsymptoms, and was more intense in the clinically affecteddogs than in asymptomatic and control dogs (p = 0.006,Kruskal Wallis and Student-Newman-Keuls tests) (Figure2B and Additional file 1). Similar results have been previ-ously observed in Leishmania infantum-infected dogs[10]. Although skin inflammation is a common finding indogs because of sandfly bites, traumas or other parasiticinfections, our data showing minimal intensity of inflam-matory infiltrate in the absence of amastigotes in the skinof asymptomatic and control dogs, and a correlationbetween the number of amastigotes and the intensity ofthe inflammatory infiltrate (R = 0.64; P < 0.05, Spearmantest) (Figure 2C) suggest that, in clinically affected dogs,the lesions were likely related to VL [17-19].

In clinically affected dogs, when few or none Leishmaniaamastigotes were observed (Figure 1C), the inflammatoryinfiltrate was constituted mainly by lymphocytes andmacrophages (Figure 1D). When many parasites werepresent (Figure 1E), the infiltrate was also comprised oflymphocytes and macrophages, as well as a larger quantityof polymorphonuclear neutrophils (PMNs) (Figure 1F).In two of these dogs (animals 5 and 7, Additional file 2),the inflammatory pattern constituted by the monomor-phic macrophage infiltrate was associated with high para-site load (Fig 1E) as observed in a study by dos Santos etal. [17]. The intense inflammatory infiltrate constituted bylymphocytes, macrophages and PMN associated withhigh parasite load in clinically affected dogs differs fromthe histopathological pattern seen in tegumentar leishma-niasis, which is illustrated by defective cell-mediatedimmunity in which Leishmania-loaded macrophages arepredominant and lymphocytes are scarce [20]. In asymp-tomatic dogs, the inflammatory infiltrate was constitutedpredominantly by macrophages and lymphocytes, similarto that observed in control dogs. The characteristics of theinflammatory infiltrate in clinically affected dogs with lowparasite burden and the absence of amastigotes in asymp-tomatic dogs suggest that these animals are immune com-petent and therefore able to control the infection for longtime.

A granulomatous inflammatory infiltrate was alsoobserved in at least one of the regions analyzed in tensymptomatic and four asymptomatic dogs. Since the pres-ence of a granulomatous lesion was associated with low orno parasites in the skin in four asymptomatic dogs, it sug-gests protective role in the tissue. Granulomatous reactionin the control of Leishmania infection has been studiedthoroughly in Leishmania donovani-infected BALB/c mousestrains and also in L. donovani-infected hamster [21-23].The control of the infection by granulomatous reaction

depends on the cytokines secreted by inflammatory cells,mainly interferon-γ and IL 2 [24].

A parameter from the inflammatory process in the skin,the presence of PMN, seems relevant for the presence ofhigher parasite load. This parameter may be analyzed insmears obtained by scarification of the ear for a quickdiagnostic approach.

Although the presence of Leishmania in the skin of the VLdog is considered to be important for the transmission ofthe parasite to the sandfly vector, it is not clear whetherthe parasites in the skin themselves are transmitted to theinsect or if they are transmitted from the capillary bloodto the sandfly during blood meal. If the latter is the case,the presence of parasites in the skin and the more intenseinflammatory infiltrate observed are just an indicationthat the parasites are reaching the skin in great amountsthrough capillary flow. The results from the present studydo not clarify this point, but suggest that the latter hypoth-esis is likely since, among symptomatic dogs, xenodiagno-sis was positive when lymphadenopathy and/orsplenomegaly were present, conditions probably relatedto higher parasitaemia and allowing for easy transmissionto the insect. A study showing no correlation of the pres-ence of Leishmania product in the skin and transmissionpotential to the vector also reinforces the latter hypothesis[4].

ConclusionThe results of the present study analyzing clinical manifes-tations, skin inflammatory processes, parasite load in theskin and transmission potential to the sandfly vector(xenodiagnosis) clarified parameters that may identifydogs that represent an immediate risk for the transmissionof Leishmania in endemic areas. These clinical manifesta-tions can be detected easily, including the presence ofPMNs in inflammatory processes of the skin. Five clinicalsigns to be considered together as strongly suggestive ofVL are abnormal nails, skin lesions, conjunctivitis, lym-phadenopathy, and weight loss. Lymphadenopathy, inparticular, was a positive clinical hallmark since it wasclosely related to the positive xenodiagnosis. Targetinganimals with these clinical signs to perform serologicaltests and parasitological exams would accelerate the iden-tification of animals with actual potential risk for trans-mission, contributing to VL transmission control inendemic areas.

Authors' contributionsBLAV participated in sample harvest, performance ofassays, data analysis and manuscript preparation. HG par-ticipated in the development of the study including anal-ysis of the data and manuscript revision. CML participatedin sample harvest, performance of assays and data analy-

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sis. ILM, SMMSS and SMC participated in discussion ofthe project, technical support and contributed to the man-uscript preparation. FALC conceived of the study andcoordinated all activity of the present study, from sampleharvest, performance of different assays, analysis of dataand manuscript preparation. All authors read andapproved of the final manuscript.

Additional material

AcknowledgementsThe authors would like to thank Mr. Manoel de Jesus Gomes da Silva for his technical contribution in preparing histopathological and immunohisto-chemical samples.

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Additional file 1Table 1. Clinical signals, xenodiagonosis and inflammatory infiltrate in 23 Leishmania (L.) chagasi-naturally infected serologically and parasi-tologically positive dogs, Xeno = xenodiagnosis; Ly = lymphocyte; Mf = macrophage; PMN = polymorphonuclear; ND = not doneClick here for file[http://www.biomedcentral.com/content/supplementary/1746-6148-4-45-S1.doc]

Additional file 2Table 2. Semi-quantitative analysis of amastigotes in skin of Leishmania (L.) chagasi-naturally infected dogs. ImunoperoxidaseClick here for file[http://www.biomedcentral.com/content/supplementary/1746-6148-4-45-S2.doc]

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